37 For each liquid, both the left and right sides of two drops (on different locations) were obtained for all specimens, and the average was calculated.

The specimens were packed in sealed sterile plastic bags with sterile distilled water and ultrasonicated for 20 min. Then all specimen surfaces were exposed to ultraviolet light in a laminar flow chamber for 20 min for sterilization.38 C. albicans adhesion was evaluated for all specimens, both saliva conditioned and unconditioned. For the preparation screening assay of the inoculum, the yeast C. albicans ATCC 90028 was seeded in an agar YEPD culture medium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) and incubated for 48 h at 37 °C. After this period, two loops of the cultivated yeast were transferred to 20 mL of the YNB (yeast nitrogen base) medium (Difco, Detroit, MI, USA) with 50 mM glucose. After incubation for 21 h at 37 °C, the cells were washed twice with sterile phosphate-buffered saline solution (PBS) (pH http://www.selleckchem.com/products/ABT-737.html 7.2) by agitation and centrifugation at 5000 × g for 5 min. After washing, the cells were re-suspended in 20 mL of YNB broth with 100 mM sterile glucose. C. albicans suspensions were standardized to a concentration of 1 × 107 cell/mL, spectrophotometrically. An aliquot of 3 mL of the standardized C. albicans suspension was added to each well of a 12-well microplate containing

the specimens and maintained for 90 min at 37 °C in the adhesion phase. 39 Thereafter, the specimens were carefully washed twice with 3 mL of PBS to remove the non-adhered cells. Negative PR-171 in vivo controls were sterile specimens immersed in YNB broth supplemented

with glucose at 100 mM. All experiments were performed in triplicate on three different occasions. The viability of the C. albicans cells adhering to acrylic specimen surfaces was evaluated by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide)-reduction assay, which measures the cell metabolic activity. Although XTT is a semi-quantitative colorimetric assay, 40 it correlates well with other quantitative techniques such as ATP and CFU assays 40 and 41 and, thus, it has been widely used to evaluate fungal adhesion and biofilm formation. 33 and 40 The XTT solution (Sigma Chemical Co., St. Louis, MO, USA) was prepared using ultra pure water at a concentration of 1 mg/mL, sterilized by filtration and maintained at −70 °C. The menadione solution (Sigma Chemical Co., St. Louis, MO, USA) was prepared in 0.4 mM acetone immediately before each experiment. After washing, the specimens were transferred to 12-well microplates containing, in each well, 2370 μL of PBS supplemented with 200 mM glucose, 600 μL of XTT and 30 μL of menadione. The plates were incubated in the dark for 3 h at 37 °C. The entire contents of each well were transferred to individual tubes and centrifuged at 5000 × g for 2 min.